Pmd19 t

An RNAprep pure Tissue Kit (Tiangen, Beijing, China) was used to extract total RNA from PSCs, and cDNA was subsequently synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Specific primers (Additional file 1: Table S1) were designed based on the full-length coding sequences of the four EgANXBs. After PCR, the products were extracted from the gel and cloned into vector pMD19-T (Takara, Dalian, China) for sequencing. Then, the pMD19-T-EgANXBs plasmids were subcloned into vector pET-32a ( +) (Invitrogen, Waltham, MA, USA) and transformed into Escherichia coli BL21 or Rosetta (DE3) cells (Tiangen), followed by induction with 0.24 mg/ml isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 ℃ for 12 h. The recombinant (r)EgANXBs were purified using a Ni²⁺ affinity chromatography column (Bio-Rad, Hercules, CA, USA). The imidazole was removed, and the concentration of each protein was determined using a bicinchoninic acid (BCA) protein quantification kit (Bestbio). The purified proteins (0.5 mg/well) were assessed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were stained with coomassie blue.